Cytokine Interferon‐γ suppresses the function of capsule myofibroblasts and induces cell apoptosis

Myofibroblasts (MFs), a contractile subset of fibroblasts, play a pivotal role in physiological wound healing and in the development of many fibroconnective disorders. The complex cytokine network regulating the function of MFs in joint stiffness is still poorly understood. In this in vitro study, we investigated the effect of the cytokine Interferon‐gamma (IFN‐γ) on MFs isolated from human joint capsules. MFs were cultivated either in the presence of increasing concentrations of IFN‐γ alone or in combination with IFN‐γ neutralizing antibodies. Cell viability, cytotoxicity, apoptosis, and mRNA gene expression of the MF markers alpha‐smooth muscle actin (α‐SMA) and collagen type I were analyzed in MF cultures. Contraction potential was analyzed in an established collagen gel contraction assay simulating the extracellular matrix. Using immunofluorescence staining, we could verify that MFs express IFN‐γ‐receptor (R)‐1 on their membrane. IFN‐γ decreased MF viability and significantly elevated the apoptosis rate in a dose‐dependent manner. IFN‐γ down‐regulated α‐SMA and collagen type I mRNA expression which was associated with a diminished MF mediated contraction of the gel matrices. These effects were suppressed by simultaneous treatment of cells with a neutralizing IFN‐γ antibody. Our experiments confirm the hypothesis that the cytokine IFN‐γ is a crucial component of the regulatory network of capsule MFs. IFN‐γ notably influences the ability of MFs to contract collagen matrices by suppressing α‐SMA gene expression. IFN‐γ is toxic for MFs in high concentrations and may negatively regulate the number of pro‐fibrotic MFs during the healing process via induction of cell apoptosis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2524–2533, 2017.

     

Normal and keloid fibroblasts are differentially influenced by IFN‐γ and triamcinolone as well as by their combination

Impaired wound healing as well as imbalanced cell proliferation and extracellular matrix synthesis and degeneration can cause aberrant scarring. The most severe impacts of such scarring on patients' lives are stigmatization and physical restriction. Although, a broad variety of combinatorial approaches with, e.g., glucocorticoids, chemotherapeutics, and immunomodulators are used, there is still a high recurrence rate of keloids. The aim of this study was to investigate which influence interferon γ (IFN‐γ, 1.000–10.000 IU/mL) and/or triamcinolone acetonide (TA, 1 μg/mL) have on proliferation, cell viability, collagen type I synthesis, and cytokine secretion in healthy and keloid fibroblasts. It was shown that mono‐treatment with IFN‐γ or TA for 2 days induced a severe reduction of the proliferative potential in both cell species. The combinatory treatment (IFN‐γ plus TA) of keloid fibroblasts enhanced the anti‐proliferative effect of the mono‐treatments, whereas no additional anti‐proliferative effect was observed in normal fibroblasts. Furthermore, we observed that the combinatory treatment regimen reduced the expression of α‐smooth muscle actin (α‐SMA), an actin isotype contributing to cell‐generated mechanical tension, in keloid fibroblasts. In normal fibroblasts, α‐SMA was reduced by the mono‐treatment with IFN‐γ as well as by the combinatory treatment. The analysis of collagen‐type I synthesis revealed that TA did not reduce collagen type I synthesis in normal fibroblasts but in keloid fibroblasts. IFN‐γ reduced in both cell species the collagen type I synthesis. The combination of TA and IFN‐γ intensified the previously observed collagen type I synthesis reduction in keloid fibroblasts. The herein presented data suggest the combinatory application of IFN‐γ and TA as a promising therapy concept for keloids.     

Simvastatin inhibits transforming growth factor‐β1‐induced expression of type I collagen,CTGF, and α‐SMA in keloid fibroblasts

Simvastatin, a 3‐hydroxy‐3‐methylglutaryl coenzyme‐A reductase inhibitor, is used to reduce cholesterol levels. Accumulating evidence has revealed the immunomodulatory and anti‐inflammatory effects of simvastatin that prevent cardiovascular diseases. In addition, the beneficial effects of statins on fibrosis of various organs have been reported. However, the functional effect of statins on dermal fibrosis of keloids has not yet been explored. The objective of this study was to determine whether simvastatin could affect dermal fibrosis associated with keloids. We examined the effect of simvastatin on transforming growth factor (TGF)‐β1‐induced production of type I collagen, connective tissue growth factor (CTGF or CCN2), and α‐smooth muscle actin (α‐SMA). Keloid fibroblasts were cultured and exposed to different concentrations of simvastatin in the presence of TGF‐β1, and the effects of simvastatin on TGF‐β1‐induced collagen and CTGF production in keloid fibroblasts were determined. The type I collagen, CTGF, and α‐SMA expression levels and the Smad2 and Smad3 phosphorylation levels were assessed by Western blotting. The effect of simvastatin on cell viability was evaluated by assessing the colorimetric conversion of 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide. Simvastatin suppressed TGF‐β1‐induced type I collagen, CTGF, and α‐SMA production in a concentration‐dependent manner. The TGF‐β1‐induced Smad2 and Smad3 phosphorylation levels were abrogated by simvastatin pretreatment. The inhibition of type I collagen, CTGF, and α‐SMA expression by simvastatin was reversed by geranylgeranyl pyrophosphate, suggesting that the simvastatin‐induced cellular responses were due to inhibition of small GTPase Rho involvement. A RhoA activation assay showed that preincubation with simvastatin significantly blocked TGF‐β1‐induced RhoA activation. The Rho‐associated coiled kinase inhibitor Y27632 abrogated TGF‐β1‐induced production of type I collagen, CTGF, and α‐SMA. However, Y27632 had no significant effect on TGF‐β1‐induced phosphorylation of Smad2 and Smad3. In conclusion, the present study suggests that simvastatin is an effective inhibitor of TGF‐β1‐induced type I collagen, CTGF, and α‐SMA production in keloid fibroblasts.     

Auxiliary proteins that facilitate formation of collagen‐rich deposits in the posterior knee capsule in a rabbit‐based joint contracture model

Post‐traumatic joint contracture is a debilitating consequence of trauma or surgical procedures. It is associated with fibrosis that develops regardless of the nature of initial trauma and results from complex biological processes associated with inflammation and cell activation. These processes accelerate production of structural elements of the extracellular matrix, particularly collagen fibrils. Although the increased production of collagenous proteins has been demonstrated in tissues of contracted joints, researchers have not yet determined the complex protein machinery needed for the biosynthesis of collagen molecules and for their assembly into fibrils. Consequently, the purpose of our study was to investigate key enzymes and protein chaperones needed to produce collagen‐rich deposits. Using a rabbit model of joint contracture, our biochemical and histological assays indicated changes in the expression patterns of heat shock protein 47 and the α‐subunit of prolyl 4‐hydroxylase, key proteins in processing nascent collagen chains. Moreover, our study shows that the abnormal organization of collagen fibrils in the posterior capsules of injured knees, rather than excessive formation of fibril‐stabilizing cross‐links, may be a key reason for observed changes in the mechanical characteristics of injured joints. This result sheds new light on pathomechanisms of joint contraction, and identifies potentially attractive anti‐fibrotic targets. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:489–501, 2016.     

Anti‐inflammatory effect of platelet‐rich plasma on nucleus pulposus cells with response of TNF‐α and IL‐1

The purpose of this study was to investigate the anti‐inflammatory effect of platelet‐rich plasma (PRP) with collagen matrix on human nucleus pulposus (NP) cell in response to pro‐inflammatory cytokines such as tumor necrosis factor‐alpha (TNF‐α) and interleukin‐1 (IL‐1). NP cells from human disks were cultured in a monolayer and maintained in the collagen matrix prior to the addition of recombinant human IL‐1 and TNF‐α. After applying IL‐1 and TNF‐α, PRP prepared by using a commercially available platelet concentration system was added. The response was investigated using real‐time PCR for mRNA expression of type II collagen, aggrecan, matrix metalloproteinase‐3 (MMP‐3), and cyclooxygenase‐2 (COX‐2). The combination of IL‐1β and TNF‐α led to decrease of matrix synthesis gene expression such as collagen type II and aggrecan and increase of the degradation gene expression of COX‐2 and MMP‐3, compared to the control. Consecutive PRP exposure significantly recovered the down‐regulated gene expression of collagen type II and aggrecan and significantly reduced the increased MMP‐3 and COX‐2 gene expression, compared to that of control groups with pro‐inflammatory cytokines. The administration of PRP with collagen matrix markedly suppressed cytokine‐induced pro‐inflammatory degrading enzymes and mediators in the NP cell. It also rescued gene expression concerning matrix synthesis, thereby stabilizing NP cell differentiation. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:551–556, 2014.     

The influence of interleukin‐4 on ligament healing

Despite a complex cascade of cellular events to reconstruct the damaged extracellular matrix, ligament healing results in a mechanically inferior scarred ligament. During normal healing, granulation tissue expands into any residual normal ligamentous tissue (creeping substitution), resulting in a larger region of healing, greater mechanical compromise and an inefficient repair process. To control creeping substitution and possibly enhance the repair process, the antiinflammatory cytokine, interleukin‐4 (IL‐4), was administered to rats before and after rupture of their medial collateral ligaments. In vitro experiments showed a time‐dependent effect on fibroblast proliferation after IL‐4 treatment. In vivo treatments with IL‐4 (100 ng/mL IV) for 5 days resulted in decreased wound size and type III collagen and increased type I procollagen, indicating a more regenerative early healing in response to the IL‐4 treatment. However, continued treatment of IL‐4 to day 11 antagonized this early benefit and slowed healing. Together, these results suggest that IL‐4 not only influences the macrophages and T lymphocytes but also stimulates fibroblasts associated with the proliferative phase of healing in a dose‐, cell‐, and time‐dependent manner. Although treatment significantly influenced healing in the first week after injury, IL‐4 alone was unable to maintain this early regenerative response.     

Effect of pro‐inflammatory and immunoregulatory cytokines on human tenocytes

Tendon injury induces a local inflammatory response, characterized by the induction of pro‐inflammatory cytokines. The aim of the present study was to analyze the effects of TNFα, IL‐6 and IL‐10 on key parameters of tendon homeostasis. Cultured primary human tenocytes were treated with the recombinant cytokines IL‐6, IL‐10, TNFα, or combinations of TNFα with IL‐6 and IL‐10 (10 ng/mL, 6, 24 h). Expression of type I collagen, elastin, MMP‐1, TNFα, IL‐1β, IL‐6, IL‐10, and suppressors of cytokine signaling (SOCS1, 3) was analyzed with the use of RTD‐PCR, immunocytochemistry, and Western blot analysis. In response to TNFα, tenocytes reduced their type I collagen deposition but increased their elastin gene expression and highly upregulated their expression for MMP‐1, pro‐inflammatory (TNFα, IL‐1β) and immunoregulatory (IL‐6, IL‐10) cytokines. TNFα stimulation augmented SOCS1, whereas SOCS3 expression in tenocytes was also induced by IL‐6. The treatment of tenocytes with IL‐6 and IL‐10 had no effect on cytokine expression. Neither IL‐6 nor IL‐10 modulated the observed effects of TNFα significantly. These results indicate that TNFα strongly activates the tenocytes to amplify their own TNFα expression and, subsequently, that of other regulatory cytokines and matrix degrading enzymes. However, the impact of IL‐6 and IL‐10 on tenocytes remains unclear. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1071–1077, 2010     

Bovine meniscal tissue exhibits age‐ and interleukin‐1 dose‐dependent degradation patterns and composition‐function relationships

Despite increasing evidence that meniscal degeneration is an early event in the development of knee osteoarthritis, relatively little is known regarding the sequence or functional implications of cytokine–induced meniscal degradation or how degradation varies with age. This study examined dose‐dependent patterns of interleukin‐1 (IL‐1)‐induced matrix degradation in explants from the radially middle regions of juvenile and adult bovine menisci. Tissue explants were cultured for 10 days in the presence of 0, 1.25, 5, or 20 ng/ml recombinant human IL‐1α. Juvenile explants exhibited immediate and extensive sulfated glycosaminoglycan (sGAG) loss and subsequent collagen release beginning after 4–6 days, with relatively little IL‐1 dose‐dependence. Adult explants exhibited a more graded response to IL‐1, with dose‐dependent sGAG release and a lower fraction of sGAG released (but greater absolute release) than juvenile explants. In contrast to juvenile explants, adult explants exhibited minimal collagen release over the 10‐day culture. Compressive and shear moduli reflected the changes in explant composition, with substantial decreases for both ages but a greater relative decrease in juvenile tissue. Dynamic moduli exhibited stronger dependence on explant sGAG content for juvenile tissue, likely reflecting concomitant changes to both proteoglycan and collagen tissue components. The patterns of tissue degradation suggest that, like in articular cartilage, meniscal proteoglycans may partially protect collagen from cell‐mediated degeneration. A more detailed view of functional changes in meniscal tissue mechanics with degeneration will help to establish the relevance of in vitro culture models and will advance understanding of how meniscal degeneration contributes to overall joint changes in early stage osteoarthritis. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:801–811, 2016.     

Long‐term follow‐up comparison of two different bi‐layer dermal substitutes in tissue regeneration: Clinical outcomes and histological findings

Double layer dermal substitute (DS) consist of a 3‐dimensional collagen structures and a superficial silicon layer that are positioned within the defect provide to promote tissue regeneration in skin wounds. DS often have unique physical characteristics due to differences in manufacturing techniques. The aim of this study is the clinical and histological comparison of Nevelia and Integra double layer DSs in patients with post‐traumatic injury wounds. Thirty patients with post‐traumatic wounds localised on the inferior limbs were randomised in 2 groups Nevelia or Integra, followed by autologous dermal epidermal graft (DEG). Clinical results were evaluated through the healing time; Manchester Scar Scale (MSS) and Visual Analog Scale (VAS) at 1, 2, and 3 weeks and after 1 and 3 years. Histological and immunohistochemical evaluation were performed at 0, 2, and 3 weeks. The difference in healing time between groups (P = .467, log‐rank test), pain and self‐estimation was not statistically significant after 35, 42, and 49 days and at 1‐year follow up. Histological data showed evident healing of wound after 2 weeks compared with preoperative with both DSs. At 3 weeks reepithelialisation and dermal regeneration were evident with both substitutes; however Nevelia showed early regenerative properties in terms of epidermal proliferation and dermal renewal compared with Integra. Nevelia showed also a more evident angiogenesis vs Integra evaluated as α‐SMA immunohistochemistry. Differences in the MSS score were statistically significant at 3 years follow up in favour of Nevelia group (P = .001). At long‐term follow up, Nevelia showed a better clinical outcome measured as MSS score vs Integra measured as MSS. Histological and immunohistochemistry data showed that Nevelia allows faster neoangiogenesis and tissue regeneration with neoformed tissue architecture closer to the physiology of the skin.     

Expression patterns of collagen types I and III in the capsule of a rat knee contracture model

Our objective was to determine the changes in expression of collagen types I and III in the capsule of a rat knee contracture model. The unilateral knee joints of adult male rats were rigidly immobilized at 150° of flexion using a rigid plastic plate and screws for 3 days, 1, 2, 4, 8, and 16 weeks (immobilized group). Sham‐operated animals had holes drilled in the femur and tibia with screws inserted without a plate (control group). The expression patterns of collagen types I and III in the anterior and posterior capsule were evaluated by in situ hybridization (ISH), quantitative real‐time polymerase chain reaction (qPCR), immunohistochemistry (IHC), and Western blotting (WB). Expressions of collagen types I and III were decreased after immobilization compared to the control group by ISH and qPCR. The expression was not changed after immobilization compared to the control group by IHC and WB. The expression of mRNA and protein levels of collagen types I and III were not increased after immobilization, which indicated that accumulation of the two types of collagen was not the etiology of joint contracture. Another process, such as capsule and synovial adhesions, may be one possible cause of joint contracture. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:315–321, 2010     

祛风止痛胶囊对关节炎模型大鼠抗炎作用及机制的实验研究

目的：研究中药祛风止痛胶囊对关节炎模型大鼠的关节炎症、肿胀、结构破坏的影响,探讨其抗炎作用机制。方法：50只雄性SD大鼠随机分为5组：正常对照组,模型组,祛风止痛胶囊低、中、高剂量组,每组10只。除正常对照组外,其他4组应用Ⅱ型胶原-弗氏完全佐剂诱导大鼠关节炎,用祛风止痛胶囊连续灌胃给药2周进行治疗,测量大鼠后肢足爪体积,观察大鼠踝关节组织病理学变化,并用ELISA方法检测大鼠血清中白细胞介素（IL）10、肿瘤坏死因子（TNF）α的含量。结果：与模型组比较,祛风止痛胶囊降低了大鼠后足爪肿胀程度。大鼠踝关节组织病理学变化显示,祛风止痛胶囊减轻滑膜组织的异常增生,炎性细胞浸润呈剂量依赖性的降低;大鼠血清中促炎细胞因子TNFα的水平变化不明显,而抑制炎症的细胞因子IL10水平明显升高。结论：中药祛风止痛胶囊通过上调抑制炎症的细胞因子IL10的水平而抑制了大鼠关节炎的滑膜增生和软骨的破坏,提示祛风止痛胶囊可作为类风湿关节炎的治疗药物。     

Sulforaphane suppresses cell growth and collagen expression of keloid fibroblasts

Keloids are fibroproliferative diseases characterized by the accumulation of an extracellular matrix including collagen. Various growth factors, or cytokines, and their receptors are overexpressed in keloids, and they are expected to be therapy targets. Sulforaphane, a dietary isothiocyanate, has recently shown anti‐tumor, anti‐inflammatory, and anti‐fibrotic properties. In this study, we found that sulforaphane inhibited cell growth and reduced collagen at the mRNA and protein levels in keloid fibroblasts. Moreover, sulforaphane markedly suppressed the expression of IL‐6 and α‐SMA and inhibited Stat3 and Smad3 signaling pathways in keloid fibroblast KF112 cells. Sulforaphane induced G2/M cell‐cycle arrest with the induction of p21 in KF112 cells. In addition, sulforaphane inhibited cell growth and suppressed the expression of collagen in keloid fibroblasts under a coculture with peripheral blood mononuclear cells. Furthermore, sulforaphane suppressed IL‐6, Stat3, and Smad3 signaling in the coculture system. This study suggests that sulforaphane may be a novel keloid treatment.     

Development of arthrogenic joint contracture as a result of pathological changes in remobilized rat knees

This study aimed to elucidate how rats recover from immobilization‐induced knee joint contracture. Rats’ right knees were immobilized by an external fixator at a flexion of 140° for 3 weeks. After removal of the fixator, the joints were allowed to move freely (remobilization) for 0, 1, 3, 7, or 14 days (n = 5 each). To distinguish myogenic and arthrogenic contractures, the passive extension range of motion was measured before and after myotomy of the knee flexors. Knee joints were histologically analyzed and the expression of genes encoding inflammatory or fibrosis‐related mediators, interleukin‐1β (1L‐1β ), fibrosis‐related transforming growth factor‐β1 (TGF‐β1 ), and collagen type I (COL1A1 ) and III (COL3A1 ), were examined in the knee joint posterior capsules using real‐time PCR. Both myogenic and arthrogenic contractures were established within 3 weeks of immobilization. During remobilization, the myogenic contracture decreased over time. In contrast, the arthrogenic contracture developed further during the remobilization period. On day 1 of remobilization, inflammatory changes characterized by edema, inflammatory cell infiltration, and upregulation of IL‐1β gene started in the knee joint posterior capsule. In addition, collagen deposition accompanied by fibroblast proliferation, with upregulation of TGF‐β1 , COL1A1 , and COL3A1 genes, appeared in the joint capsule between days 7 and 14. These results suggest the progression of arthrogenic contracture following remobilization, which is characterized by fibrosis development, is possibly triggered by inflammation in the joint capsule. It is therefore necessary to focus on developing new treatment strategies for immobilization‐induced joint contracture. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1414–1423, 2017.

     

Discovery of the natural anti‐Gal antibody and its past and future relevance to medicine

This is a personal account of the discovery of the natural anti‐Gal antibody, the most abundant natural antibody in humans, the reciprocal distribution of this antibody and its ligand the α‐gal epitope in mammals and the immunological barrier this antibody has formed in porcine to human xenotransplantation. This barrier has been overcome in the recent decade with the generation of α1,3‐galactosyltransferase gene‐knockout pigs. However, anti‐Gal continues to be relevant in medicine as it can be harnessed for various therapeutic effects. Anti‐Gal converts tumor lesions injected with α‐gal glycolipids into vaccines that elicit a protective anti‐tumor immune response by in situ targeting of tumor cells for uptake by antigen‐presenting cells. This antibody further accelerates wound and burn healing by interaction with α‐gal nanoparticles applied to injured areas and induction of rapid recruitment and activation of macrophages. Anti‐Gal/α‐gal nanoparticle immune complexes may further induce rapid recruitment and activation of macrophages in ischemic myocardium and injured nerves, thereby inducing tissue regeneration and prevention of fibrosis.     

Increased IL‐1β expression and myofibroblast recruitment in subacromial bursa is associated with rotator cuff lesions with shoulder stiffness

We evaluated whether proinflammatory cytokine expression and myofibroblast recruitment in subacromial bursa was linked to rotator cuff lesions with shoulder stiffness. We analyzed expressions of IL‐1β, IL‐6, and TNF‐α in subacromial bursa and joint fluid collected from 14 patients with cuff tears with stiffness as a study group (Group I) and 14 patients with rotator cuff tears without shoulder stiffness as a control group (Group II) using real‐time RT‐PCR, immunohistochemistry, and ELISA. Myofibroblast apoptosis in subacromial bursa was analyzed using terminal deoxynucleotidyl transferase ‐mediated deoxyuridine triphosphate‐biotin nick end‐labeling (TUNEL) and α‐smooth muscle actin immunofluorescence staining. Shoulder function was evaluated using the Constant score. Group I had higher mRNA expression (p < 0.001) and immunoreactivities (p < 0.001) of IL‐1β. They also had higher levels of IL‐1β, IL‐6, and TNF‐α in joint fluid. Increased IL‐1β mRNA expression in the subacromial bursa and IL‐1β levels in joint fluid were correlated with a preoperative deficit in shoulder motion (p < 0.001) and preoperative Constant scores (p < 0.001). Immunofluorescence observations showed that Group I subjects had more myofibroblasts (p < 0.001) than Group II. In Group II, a significant correlation was found between apoptotic myofibroblasts and total myofibroblasts (p = 0.002), but not in Group I (p = 0.510). Increased expression of IL‐1β and myofibroblast recruitment in the subacromial bursa in rotator cuff lesions are linked to shoulder stiffness. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1090–1097, 2008     

Pro‐healing effects of bilirubin in open excision wound model in rats

Bilirubin, a by‐product of heme degradation, has an important role in cellular protection. Therefore, we speculated that bilirubin could be of potential therapeutic value in wound healing. To validate the hypothesis, we used a full‐thickness cutaneous wound model in rats. Bilirubin (30 mg/kg) was administered intraperitoneally every day for 9 days. The surface area of the wound was measured on days 0, 2, 4, 7 and 10 after the creation of the wound. The granulation tissue was collected on day 10 post‐wounding for analysing various parameters of wound healing. Bilirubin treatment accelerated wound contraction and increased hydroxyproline and glucosamine contents. mRNA expression of pro‐inflammatory factors such as intercellular cell adhesion molecule‐1 (ICAM‐1) and tumour necrosis factor‐α (TNF‐α) were down‐regulated and that of anti‐inflammatory cytokine interleukin‐10 (IL‐10) was up‐regulated. The findings suggest that bilirubin could be a new agent for enhancing cutaneous wound healing.     

Rosiglitazone treatment attenuates renal tissue inflammation generated by urinary tract obstruction

Aim: Peroxisome proliferator‐activated receptor (PPAR)‐γ activation by rosiglitazone decreases manifestation of intrarenal inflammatory hallmarks. Inflammation significantly aggravates renal injury following urinary tract obstruction. The effect of rosiglitazone on renal inflammation following unilateral ureteral obstruction was investigated. Methods: Ninety‐six Srague–Dawley rats were subjected to unilateral ureteral ligation, or to sham operation. Half of each group received rosiglitazone, 5 mg/kg bodyweight per day. The animals were killed and their kidneys allocated following 1 h, 24 h or 2 weeks, for pathological examination or for intrarenal transforming growth factor (TGF)‐β, interleukin (IL)‐4, IL‐6, IL‐10 and nitric oxide (NO) assessment by specific enzyme‐linked immunosorbent assays. Apoptosis rates, extracellular matrix deposition, PPAR‐γ, α‐smooth muscle actin (α‐SMA) expression and macrophage infiltration were assessed by specific immunohistological stainings. Results: PPAR‐γ receptor expression was downregulated, and infiltration of macrophages decreased, in all rosiglitazone‐treated kidneys. Rosiglitazone significantly decreased apoptosis, TGF‐β, IL‐6, α‐SMA expression and NO availability in obstructed kidneys. Synthesis of IL‐10 was unaltered, while IL‐4 augmented by Rosiglitazone. Rosiglitazone also affected NO and IL‐4 production in sham‐operated controls. Conclusion: (i) Rosiglitazone attenuates profibrotic and pro‐inflammatory responses in a rat model of ureteral obstruction‐induced renal inflammation; (ii) rosiglitazone stimulates counteractive anti‐inflammatory responses in the damaged kidneys; (iii) in part, rosiglitazone exerts comparable anti‐inflammatory effects on obstructed kidneys and unobstructed healthy controls. Taken together, this ascertains the importance of the anti‐inflammatory role of rosiglitazone treatment in amelioration of ureteral obstruction‐induced renal damage.     

Platelet‐rich plasma (PRP) impairs the craniofacial bone repair associated with its elevated TGF‐β levels and modulates the co‐expression between collagen III and α‐smooth muscle actin

Transforming growth factor‐β (TGF‐β) is considered the main inducer of both the α‐smooth muscle actin (α‐SMA) phenotype and collagen synthesis and deposition and plays a significant role in the tissue repair and the development of fibrosis. Since the PRP constitutes an important source of TGF‐β and its efficacy on the craniofacial bone repair remains controversy, the aim of this study was to evaluate the effect of PRP in the presence of levels of TGF‐β on PRP samples, as well as in the presence of collagen III and α‐SMA+ cells, while comparing these results by means of a histomorphometric analysis of the bone matrix and fibrous deposition on the bone repair. Four bone defects of 16 mm² were created on the calvarium of 21 rabbits. The surgical defects were treated with either particulate autograft, particulate autograft mixed with PRP and PRP alone. Animals were euthanized at 15, 30, and 45 days postoperative. Histomorphometric and immunohistochemical analyses were performed to assess repair time, as well as the expression of collagen III, and α‐SMA. The histomorphometric results demonstrated intensive deposition of fibrous tissue while hinder bone deposition occurred in PRP groups. These results coincided with higher values of the TGF‐β on the PRP sample, also larger occurrence of diffuse collagen III deposition and higher presence of α‐SMA+ cells spread among the fibrous tissue. Thus, the higher levels of TGF‐β associated with the both expression of collagen III and α‐SMA on defect treated with PRP suggest that its biomaterial induce an effect that can be considered similarly to a fibroproliferative disorder. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:457–463, 2011     

Seasonal immunohistochemical reactivity of S‐100 and α‐smooth muscle actin proteins in the epididymis of dromedary camel,Camelus dromedarius

The S‐100 and alpha smooth muscle actin (α‐SMA) proteins have been localised in epididymal tissue of several mammalian species, but there have been no data for a seasonal work in camel. The aim of this study was to investigate the immunoreactivities of S‐100 and α‐SMA proteins in the epididymis of dromedary camel during breeding and nonbreeding seasons. The immunopositive signals for both proteins were observed in different regions of camel epididymis. S‐100‐immunopositive signals were noted in both the epididymal epithelium and the intertubular connective tissue, while α‐SMA signals were confined to the intertubular connective tissue, especially in the peritubular smooth muscle coat and the blood vessels. This study showed an increase in the intensity of S‐100 and α‐SMA immunoreactions during the breeding season in different regions of camel epididymis than that seen in the nonbreeding season. In conclusion, epididymis might be considered as a source of S‐100 and α‐SMA proteins in the camel and the secretion of these proteins showed distinct seasonal variations. Further, S‐100 and α‐SMA may affect the structural and physiological states of the epididymal duct.